Activation of c-myc Gene Expression by Tumor-Derived p53 Mutants Requires a Discrete C-Terminal Domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3′ end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

Activation of Phospholipase C β by Gβγ and Gαq Involves C-Terminal Rearrangement to Release Autoinhibition

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Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A

An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K_m of the NADH oxidase after activation by lipids was four- to fivefold less than the K_m before activation. The V_max was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A₂ block the stimulation of the oxidase by auxins.     

Calcium-Induced Folding of a Beta Roll Motif Requires C-Terminal Entropic Stabilization

Beta roll motifs are associated with several proteins secreted by the type 1 secretion system (T1SS). Located just upstream of the C-terminal T1SS secretion signal, they are believed to act as calcium-induced switches that prevent folding before secretion. Bordetella pertussis adenylate cyclase (CyaA) toxin has five blocks of beta roll motifs (or repeats-in-toxin motifs) separated by linkers. The block V motif on its own has been reported to be non-responsive to calcium. Only when the N- and C-terminal linkers, or flanking groups, were fused did the motif bind calcium and fold. In an effort to understand the requirements for beta roll folding, we have truncated the N- and C-terminal flanks at several locations to determine the minimal essential sequences. Calcium-responsive beta roll folding occurred even in the absence of the natural N-terminal flank. The natural C-terminal flank could not be truncated without decreased calcium affinity and only partially truncated before losing calcium-responsiveness. Globular protein fusion at the C-terminus likewise enabled calcium-induced folding but fusions solely at the N-terminus failed. This demonstrates that calcium-induced folding is an inherent property of the beta roll motif rather than the flanking groups. Given the disparate nature of the observed functional flanking groups, C-terminal fusions appear to confer calcium-responsiveness to the beta roll motif via a non-specific mechanism, suggesting that entropic stabilization of the unstructured C-terminus can enable beta roll folding. Increased calcium affinity was observed when the natural C-terminal flank was used to enable calcium-induced folding, pointing to its cooperative participation in beta roll formation. This work indicates that a general principle of C-terminal entropic stabilization can enable stimulus-responsive repeat protein folding, while the C-terminal flank has a specific role in tuning calcium-responsive beta roll formation. These observations are in stark contrast to what has been reported for other repeat proteins.     

Identification of the Major Ubiquitin-binding Domain of the Pseudomonas aeruginosa ExoU A2 Phospholipase

Numerous Gram-negative bacterial pathogens use type III secretion systems to deliver effector molecules into the cytoplasm of a host cell. Many of these effectors have evolved to manipulate the host ubiquitin system to alter host cell physiology or the location, stability, or function of the effector itself. ExoU is a potent A₂ phospholipase used by Pseudomonas aeruginosa to destroy membranes of infected cells. The enzyme is held in an inactive state inside of the bacterium due to the absence of a required eukaryotic activator, which was recently identified as ubiquitin. This study sought to identify the region of ExoU required to mediate this interaction and determine the properties of ubiquitin important for binding, ExoU activation, or both. Biochemical and biophysical approaches were used to map the ubiquitin-binding domain to a C-terminal four-helix bundle of ExoU. The hydrophobic patch of ubiquitin is required for full binding affinity and activation. Binding and activation were uncoupled by introducing an L8R substitution in ubiquitin. Purified L8R demonstrated a parental binding phenotype to ExoU but did not activate the phospholipase in vitro. Utilizing these new biochemical data and intermolecular distance measurements by double electron-electron resonance, we propose a model for an ExoU-monoubiquitin complex.     

Alteration of Phospholipase C-δ Protein Level and Specific Activity in Alzheimer's Disease

Abstract: Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. We have previously demonstrated that an antibody to an isozyme of PLC, PLC-δ, produced intense staining of neurofibrillary tangles in the brains of patients with Alzheimer's disease. In the present study, we investigated the protein level and activity of this enzyme in control and Alzheimer brains. Western blot analysis using a specific antibody for PLC-δ showed that the concentration of PLC-δ protein was significantly higher in the cytosolic fraction of Alzheimer's disease cortical tissue than in control brains. The activity of PLC-δ, which hydrolyzes phosphatidylinositol, was also investigated, and we found that PLC-δ activity was not significantly different in the Alzheimer and control cytosolic fractions. These results indicate that the specific activity of PLC-δ is decreased in Alzheimer brains and suggest that inactivation of PLC-δ might be related to the pathophysiology of this disease.     

Multiple Domains Are Required for the Toxic Activity of Pseudomonas aeruginosa ExoU

Expression of ExoU by Pseudomonas aeruginosa is correlated with acute cytotoxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or significant homology with other proteins suggests that ExoU may possess a new mechanism of toxicity. To study the intracellular effects of ExoU, we developed a transient-transfection system in Chinese hamster ovary cells. Transfection with full-length but not truncated forms of ExoU inhibited reporter gene expression. Inhibition of reporter activity after cotransfection with ExoU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpression of the remainder of the molecule from separate plasmids in trans. This strategy was used to map N- and C-terminal regions of ExoU that are necessary but not sufficient for toxicity. Disruption of a middle region of the protein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In contrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligomerize or form aggregates. The complex domain structure of ExoU suggests that, like other P. aeruginosa-encoded type III effectors (ExoS and ExoT), ExoU toxicity may result from a molecule that possesses more than one activity.     

A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties

The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physiochemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol-rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes composed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane.     

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4) (reviewed in [1-4]). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood [5, 6]. The?Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain [7, 8] whose primary function is to bind the 3' end of small RNAs [9-13]. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells.?We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs,?PAZ-disrupted Agos bound duplex small interfering RNAs,?but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics.     

Activation of Phospholipase A by Plant Defense Elicitors

Participation of phospholipase A (PLase A) in plant signal transduction has been documented for auxin stimulation of growth but not for elicitation of any plant defense response. In this paper, we report two independent assays for monitoring PLase A induction in plant cells and have used these assays to evaluate whether transduction of defense-related signals might require PLase A activation. Oligogalacturonic acid, a potent elicitor of the soybean (Glycine max) H2O2 burst, was unable to stimulate endogenous PLase A, suggesting that PLase A activation is not an obligate intermediate in the oligogalacturonic acid-induced burst pathway. In contrast, harpin and an extract from the pathogenic fungus Verticillium dahliae both stimulated the oxidative burst and promoted a rapid increase in PLase A activity. To evaluate the possible role of this inducible PLase A activity in transducing the oxidative burst, we tested the effect of chlorpromazine-HCl, a PLase A inhibitor on elicitor-stimulated burst activity. Pretreatment with chloropromazine was found to inhibit the H2O2 burst triggered by V. dahliae extract at the same concentration at which it blocked PLase A activation. In contrast, neither the harpin- nor oligogalacturonic acid-induced burst was altered by addition of chlorpromazine. These data suggest that PLase A stimulation may be important in certain elicitor-induced oxidative bursts (e.g. V. dahliae) and that other elicitors such as oligogalacturonic acid and harpin must operate through independent signaling intermediates to activate the same defense response.     

The C-Terminal Regions Have an Important Role in the Activity of the Ferroxidase Center and the Stability of Chlorobium tepidum Ferritin

The recombinant Chlorobium tepidum ferritin (rCtFtn) is able to oxidize iron using ferroxidase activity but its ferroxidase activity is intermediate between the H-chain human ferritin and the L-chain human ferritin. The rCtFtn has an unusual C-terminal region composed of 12 histidine residues, as well as aspartate and glutamate residues. These residues act as potential metal ion ligands, and the rCtFtn homology model predicts that this region projects inside the protein cage. The rCtFtn also lacks a conserved Tyr residue in position 19. In order to know if those differences are responsible for the altered ferroxidase properties of rCtFtn, we introduced by site-directed mutagenesis a stop codon at position 166 and a Tyr residue replaced Ala19 in the gene of rCtFtn (rCtFtn 166). The rCtFtn166 keeps the canonical sequence considered important for the activity of this family of proteins. Therefore, we expected that rCtFtn 166 would possess similar properties to those described for this protein family. The rCtFtn 166 is able to bind, oxidize and store iron; and its activity is inhibit by Zn(II) as was described for other ferritins. However, the rCtFtn 166 possesses a decrease ferroxidase activity and protein stability compared with the wild type rCtFtn. The analysis of the Ala19Tyr rCtFtn shows that this change does not affect the kinetic of iron oxidation. Therefore, these results indicate that the C-terminal regions have an important role in the activity of the ferroxidase center and the stability of rCtFtn.     

Activation of Protein Kinase PKR Requires Dimerization-induced cis-Phosphorylation within the Activation Loop

Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKR^T446∼P-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKR^T446∼P-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.     

PKC-Dependent Human Monocyte Adhesion Requires AMPK and Syk Activation

PKC plays a pivotal role in mediating monocyte adhesion; however, the underlying mechanisms of PKC-mediated cell adhesion are still unclear. In this study, we elucidated the signaling network of phorbol ester PMA-stimulated human monocyte adhesion. Our results with pharmacological inhibitors suggested the involvement of AMPK, Syk, Src and ERK in PKC-dependent adhesion of THP-1 monocytes to culture plates. Biochemical analysis further confirmed the ability of PMA to activate these kinases, as well as the involvement of AMPK-Syk-Src signaling in this event. Direct protein interaction between AMPK and Syk, which requires the kinase domain of AMPK and linker region of Syk, was observed following PMA stimulation. Notably, we identified Syk as a novel downstream target of AMPK; AICAR can induce Syk phosphorylation at Ser178 and activation of this kinase. However, activation of AMPK alone, either by stimulation with AICAR or by overexpression, is not sufficient to induce monocyte adhesion. Studies further demonstrated that PKC-mediated ERK signaling independent of AMPK activation is also involved in cell adhesion. Moreover, AMPK, Syk, Src and ERK signaling were also required for PMA to induce THP-1 cell adhesion to endothelial cells as well as to induce adhesion response of human primary monocytes. Taken together, we propose a bifurcated kinase signaling pathway involved in PMA-mediated adhesion of monocytes. PKC can activate LKB1/AMPK, leading to phosphorylation and activation of Syk, and subsequent activation of Src and FAK. In addition, PKC-dependent ERK activation induces a coordinated signal for cytoskeleton rearrangement and cell adhesion. For the first time we demonstrate Syk as a novel substrate target of AMPK, and shed new light on the role of AMPK in monocyte adhesion, in addition to its well identified functions in energy homeostasis.     

Imprime PGG-Mediated Anti-Cancer Immune Activation Requires Immune Complex Formation

Imprime PGG (Imprime), an intravenously-administered, soluble β-glucan, has shown compelling efficacy in multiple phase 2 clinical trials with tumor targeting or anti-angiogenic antibodies. Mechanistically, Imprime acts as pathogen-associated molecular pattern (PAMP) directly activating innate immune effector cells, triggering a coordinated anti-cancer immune response. Herein, using whole blood from healthy human subjects, we show that Imprime-induced anti-cancer functionality is dependent on immune complex formation with naturally-occurring, anti-β glucan antibodies (ABA). The formation of Imprime-ABA complexes activates complement, primarily via the classical complement pathway, and is opsonized by iC3b. Immune complex binding depends upon Complement Receptor 3 and Fcg Receptor IIa, eliciting phenotypic activation of, and enhanced chemokine production by, neutrophils and monocytes, enabling these effector cells to kill antibody-opsonized tumor cells via the generation of reactive oxygen species and antibody-dependent cellular phagocytosis. Importantly, these innate immune cell changes were not evident in subjects with low ABA levels but could be rescued with exogenous ABA supplementation. Together, these data indicate that pre-existing ABA are essential for Imprime-mediated anti-cancer immune activation and suggest that pre-treatment ABA levels may provide a plausible patient selection biomarker to delineate patients most likely to benefit from Imprime-based therapy.     

Sprouty Proteins Inhibit Receptor-mediated Activation of Phosphatidylinositol-specific Phospholipase C

Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry–PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP₃) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP₃ at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.     

Autoproteolytic Activation of a Symbiosis-regulated Truffle Phospholipase A2

Fungal phospholipases are members of the fungal/bacterial group XIV secreted phospholipases A₂ (sPLA₂s). TbSP1, the sPLA₂ primarily addressed in this study, is up-regulated by nutrient deprivation and is preferentially expressed in the symbiotic stage of the ectomycorrhizal fungus Tuber borchii. A peculiar feature of this phospholipase and of its ortholog from the black truffle Tuber melanosporum is the presence of a 54-amino acid sequence of unknown functional significance, interposed between the signal peptide and the start of the conserved catalytic core of the enzyme. X-ray diffraction analysis of a recombinant TbSP1 form corresponding to the secreted protein previously identified in T. borchii mycelia revealed a structure comprising the five α-helices that form the phospholipase catalytic module but lacking the N-terminal 54 amino acids. This finding led to a series of functional studies that showed that TbSP1, as well as its T. melanosporum ortholog, is a self-processing pro-phospholipase A₂, whose phospholipase activity increases up to 80-fold following autoproteolytic removal of the N-terminal peptide. Proteolytic cleavage occurs within a serine-rich, intrinsically flexible region of TbSP1, does not involve the phospholipase active site, and proceeds via an intermolecular mechanism. Autoproteolytic activation, which also takes place at the surface of nutrient-starved, sPLA₂ overexpressing hyphae, may strengthen and further control the effects of phospholipase up-regulation in response to nutrient deprivation, also in the context of symbiosis establishment and mycorrhiza formation.